This project was designed to characterize the ligand-binding properties of opioid receptors in brain and peripheral tissues, using quantitative ligand binding studies. We have demonstrated and characterized at least four classes of sites, present simultaneously: mu-1 (high affinity Kd = 0.2 nM and nonselective for mu- or delta- selective enkephalins); mu-2 (lower affinity, mu selective), delta, and kappa. Three of these sites (mu-1, mu-2, delta) were characterized in 10 consecutive experiments using rigorous statistical criteria and a new form of "Kd versus Kd" bivariate graphical analysis. A new "multiligand" experimental design was used to improve sensitivity for detection of mu-1 sites. Naloxonazine shows mu and mu-1 selectivity. It can irreversibly or noncompetitively block about 50% of mu-1 sites, while it shows significant competitive effects at mu-2 and delta sites. The existence of mu-1 sites has considerable implications for interpreting binding, pharmacological and biochemical studies of opioid mediated systems. These methods are now being extended, to consider epsilon, kappa and sigma receptors, to study receptors of hypothalamus, adrenal medulla and vas deferens to categorize subtypes of kappa receptors, and to examine opioid-adrenergic interactions.